The challenges in the growing field of high-throughput molecular screening (HTS) call for highly sensitive, economical, and massively parallel detector systems. Recent technology trends include the development of array assays for parallel detection of 100-100,000 species at a time, the rapid growth in the use of DNA probes for mRNA expression monitoring and sequence detection, and the advent of protein/antibody and cell arrays. These trends, which are expected to continue for the foreseeable future, call for transducers compatible with hybridization assays and molecular binding, implementable in dense array formats, with high sensitivity to target molecules. While HTS has experienced revolutionary changes over the past decade, speed and accuracy remain among the major challenges. For example, rapidly evolving fluorescent-label based instrumentation, which has proven to be highly useful in DNA sequencing and hybridization detection, can be prone to significant detection errors resulting from the possibility of massive summation of weak false interactions, photobleaching, and autofluorescence. There are also limitations of scanning speed and spatial resolution, and therefore array density and feature number. As noted in the NIH Molecular Libraries Screening Instrumentation program announcement, there exists a critical need to bridge the large gap between today's molecular screening capabilities and the requirements for advanced HTS technology.
The growing interest in high-content HTS-based on protein or gene expression monitoring, or the measurement of target/ligand binding in massively parallel fashion, calls for efficient, high-throughput molecular screening instrumentation. Quantitation of large numbers of biomolecular analytes is most commonly achieved through separation by electrophoresis, or by use of affinity binding agents such nucleic acid hybridization probes, antibodies or aptamers. In nearly all methods other than mass spectrometry, what is actually detected is a label of some sort, rather than the biomolecular analyte itself. In most applications, the sensitivity of detection of this label (rather than, e.g., kinetics or affinity agent binding tightness), limits assay performance.
The introduction of each successive generation of molecular labels has transformed the practice of biomolecular assays. With many exceptions, the general trend has been that enzymes, which are still widely used but which now have yielded important applications to fluorescent labels, largely supplanted radioisotopes. Fluors are widely and very successfully used, but detection sensitivity and signal stability remain important limitations. Single-molecule fluorescence detection is now reliably practiced, but only with elaborate equipment unsuited to most HTS applications. More importantly, fundamental optical limitations posed by triplet blinking and photobleaching impose major limitations on the detection performance that can be practically achieved with fluorescent labels.
The application of magnetoresistive sensors to biomolecular recognition was suggested by Shieh and Ackley in 1996 (Shieh and Ackley, “Magnetoresistance-based method and apparatus for molecular detection,” U.S. Pat. No. 6,057,167) and first described in 1998 (Baselt et. al., “A biosensor based on magnetoresistance technology,” Journal/Biosens. Bioelectron., 13(7-8), pp. 731-739, 1998). In this biodetection scheme, magnetic particles are used as labels for biological agents and are detected using magnetoresistive elements, where the resistance of a magnetoresistive sensor changes in the presence of a magnetic particle.
In a typical giant magnetoresistive (GMR) sensor, the resistance depends on the mutual orientation of two magnetic layers in a bi-layer structure. Two possible resistance-sensing configurations are possible: current in-the-plane of the sensor (CIP) and current perpendicular to the plane of the sensor (CPP). The resistance is the highest when the two layers are magnetized in opposite directions and is lowest when magnetizations are aligned in the same direction. Relative resistance change (ΔR/R) values of 6-8% and 20-25% can be routinely obtained for CIP and CPP sensors, respectively, and can be measured in sub-100 nm magnetoresistive sensors to a precision of 0.1%, a signal-to-noise ratio of 100- to 250-fold (Wolf et. al., “Spintronics: A spin-based electronics vision for the future,” Journal/Science, 294(5546), pp. 1488-1495, 2001; Childress et. al., “Spin-valve and tunnel-valve structures with in situ in-stack bias,” Journal/IEEE Trans. Magn., 38(5), pp. 2286-2288, 2002). The presence of a magnetic particle disrupts the magnetic environment and under appropriate conditions may lead to a change in the sensor's resistance.
Considerable progress has been made in magnetic biosensor development (Edelstein et. al., “The BARC biosensor applied to the detection of biological warfare agents,” Journal/Biosens. Bioelectron., 14(10-11), pp. 805-813, 2000; Schotter et. al., “Comparison of a prototype magnetoresistive biosensor to standard fluorescent DNA detection,” Journal/Biosens. Bioelectron., 19(10), pp. 1149-1156, 2004) including the demonstration of bioconjugated magnetic multimicron-scale microbead detection using CIP GMR sensors and magnetic field removal (melting) of magnetic microbeads from the sensor surface for enhanced detection specificity. Magnetic “melting curves” are particularly promising as a means of improving the quality of hybridization and immunoassay data by discriminating against weak, non-specific interactions. Notably, magnetic removal curves are more compatible with heat-labile protein analytes, antibodies, cells and receptors than is thermal melting.
The limitations of magnetoresistive sensor and magnetic field source technologies have restricted the applications to the detection of magnetic microbeads (>2 μm, ca. 8000 times the mass and volume of those proposed for the present work) using relatively large magnetoresistive sensor elements. High magnetic-label-to-biomolecule size ratio can lead to reduced sensitivity, interference with biomolecular interactions, and highly multivalent, avidity-modified interactions, limiting the applicability of the technology.
Electrical and magnetic properties of larger magnetic sensor elements with dimensions of a micrometer and above are highly susceptible to small variations in sensor geometry making it necessary to significantly overdesign sensor array, for example, via introduction of select transistors, antiferromagnetic pinning layers and add-on current carrying coils to set the direction of magnetization. This limits the ability of the sensors to quantitatively analyze magnetic labels beyond distinguishing between “present” and “not present” events as well as making the sensors highly susceptible to external magnetic fields, which are necessary, for example, for the detection of superparamagnetic nanoparticles. Significantly, manipulation of superparamagnetic nanoparticles (such nanoparticles being a desirable choice for magnetic labels because of their unique magnetic properties) represents a major challenge as it requires generation of large magnetic field gradients not achievable with prior art macroscopic magnetic field sources. Moreover, quantitative magnetic field removal of magnetic labels has not been achieved. Magnetic field removal of particles held by a single biomolecular recognition interaction would be particularly informative. In addition, array feature density declines as the square of the magnetoresistive element size, reducing array density.